Methods and compositions for rna-guided genetic circuits

Abstract

Some aspects provide genetic circuits to engineer complex and adaptive cellular behaviors. Methods of controlling expression of output sequence(s) are also provided herein using genetic circuits that employ catalytically inactive endonucleases.

What is claimed is: 
     
         1 . A method of controlling expression of a first output sequence, comprising:
 introducing into a cell a genetic circuit comprising one or more polynucleotide sequences, wherein the genetic circuit comprises:   (a) a first output sequence;   (b) a first promoter/operator controlling transcription of the first output sequence;   (c) a first guide RNA targeting the first promoter/operator;   (d) a second promoter/operator controlling transcription of the first guide RNA, wherein the second promoter/operator is input-sensitive such that a first input signal is required for induction of transcription of the first guide RNA;   (e) a first catalytically inactive endonuclease that in combination with the first guide RNA binds to a sequence targeted by the first guide RNA and prevents transcription of the first output sequence;   (f) a third promoter/operator controlling transcription of the first catalytically inactive endonuclease, wherein the third promoter/operator is input-sensitive such that a second input signal is required for induction of transcription of the first catalytically inactive endonuclease; and   (g) one or more heterologous polymerases that specifically bind one or more of the first, second and/or third promoter/operator.   
     
     
         2 . The method of  claim 1 , wherein the genetic circuit further comprises a fourth promoter/operator controlling transcription of one or more second output sequence, wherein the fourth promoter/operator is input-sensitive such that a third input signal is required for induction of transcription of the one or more second output sequence. 
     
     
         3 . The method of  claim 1  or  claim 2 , wherein the genetic circuit further comprises one or more endogenous polymerases that bind one or more of the first, second, third and/or fourth promoter/operator. 
     
     
         4 . The method of  claim 2  or  claim 3 , wherein the third input signal is one or more second guide RNA encoded by the first output sequence and wherein the one or more second output sequence is one or more third guide RNA. 
     
     
         5 . The method of any one of  claims 1 - 4 , wherein at least two of the first, second and third input signals are the same. 
     
     
         6 . The method of any one of  claims 1 - 5 , wherein any of the first, second, third and/or fourth promoter/operator comprises a T7 promoter and an operator. 
     
     
         7 . The method of any one of  claims 1 - 6 , wherein the heterologous polymerase is a viral polymerase. 
     
     
         8 . The method of any one of  claims 1 - 7 , wherein the heterologous polymerase is a T7 RNA polymerase. 
     
     
         9 . The method of any one of  claims 1 - 8 , wherein the genetic circuit further comprises polynucleotide sequences encoding one or more decoy operators having the same, or substantially the same, sequence as one or more of the operator sequences of the first, second, third and/or fourth promoter/operators. 
     
     
         10 . The method of any one of  claims 1 - 9 , wherein the genetic circuit further comprises one or more polynucleotide sequences encoding one or more small RNAs (sRNAs) that binds to and sequesters the guide RNA. 
     
     
         11 . The method of any one of  claims 1 - 10 , wherein the first, second and/or third guide RNA is a nested guide RNA comprising two or more sequences that target two or more target sequences in the first, second, third and/or fourth promoter/operator, thereby causing promoter looping of the first, second, third and/or fourth promoter/operator. 
     
     
         12 . The method of any one of  claims 1 - 11 , wherein the first, second and/or third input signal is a chemical, light, a polypeptide or a mechanical force. 
     
     
         13 . The method of any one of  claims 1 - 12 , wherein the first, second and/or third input signal is isopropyl β-D-1-thiogalactopyranoside (IPTG), anhydrotetracycline (aTc), or 2,4-diacetylphloroglucinol (DAPG). 
     
     
         14 . The method of any one of  claims 1 - 13 , wherein the first and/or second output sequence encode one or more first output molecule and the one or more first output molecule in turn becomes a fourth input signal required for controlling transcription of one or more third output sequence. 
     
     
         15 . The method of  claim 14 , wherein the first second and/or third output sequence is a fourth guide RNA. 
     
     
         16 . The method of any one of  claims 1 - 15 , wherein two or more input signals control transcription in a single promoter/operator. 
     
     
         17 . The method of any one of  claims 1 - 16 , wherein the first catalytically inactive endonuclease is a RNA-guided DNA endonuclease. 
     
     
         18 . The method of any one of  claims 1 - 17 , wherein the first catalytically inactive endonuclease is a catalytically inactive clustered regularly interspaced short palindromic repeat (CRISPR) endonuclease. 
     
     
         19 . The method of any one of  claims 1 - 18 , wherein the first catalytically inactive endonuclease is catalytically inactive Cas9 or catalytically inactive Cpf1. 
     
     
         20 . The method of any one of  claims 1 - 19 , wherein the first catalytically inactive endonuclease is selected from the group consisting of dSpCas9, dSpCas9(E), dSpCas9(VRER), dSpCas9(VQR), dSpCas9(EQR), desSpCas9, dSpCas9-HF1, dSaCas9, desSaCas9, dSt1Cas9, dFnCpf1, dAsCpf1, and dLbCpf1. 
     
     
         21 . The method of any one of  claims 1 - 20 , wherein one or more nucleotide of the first, second, third and/or fourth guide RNA is mutated and the mutation of the first, second, third and/or fourth guide RNA does not decrease prevention of transcription of the first, second and/or third output sequence. 
     
     
         22 . The method of any one of  claims 1 - 21 , wherein the cell is a prokaryotic cell. 
     
     
         23 . The method of any one of  claims 1 - 22 , wherein the cell is a bacterial cell. 
     
     
         24 . The method of  claim 23 , wherein the genetic circuit in the bacterial cell is optimized for bioreactor growth. 
     
     
         25 . The method of any one of  claims 1 - 24 , wherein the cell is part of a microbiome. 
     
     
         26 . The method of any one of  claims 1 - 22 , wherein the cell is a BL21(DE3) cell. 
     
     
         27 . The method of any one of  claims 1 - 21 , wherein the cell is a eukaryotic cell. 
     
     
         28 . The method of any one of  claims 1 - 21 , wherein the cell is a plant cell. 
     
     
         29 . The method of any one of  claims 1 - 21 , wherein the cell is a human cell. 
     
     
         30 . The method of any one of  claims 1 - 29 , wherein the first, second, third and/or fourth promoter/operator is sensitive to a guide RNA. 
     
     
         31 . The method of any one of  claims 1 - 29 , wherein the first, second, third and/or fourth promoter/operator is sensitive to a chemical input. 
     
     
         32 . The method of any one of  claims 1 - 31 , wherein the first, second, third and/or fourth promoter/operator comprises a polynucleotide sequence that encodes a T7 promoter and a polynucleotide sequence that encodes a PhlF operator. 
     
     
         33 . The method of  claim 32 , wherein the T7 promoter and the PhlF operator control the transcription of guide RNA A2NT. 
     
     
         34 . The method of  claim 33 , wherein the guide RNA A2NT controls the transcription of the A2NT operator. 
     
     
         35 . The method of any one of  claims 1 - 34 , wherein the first, second and/or third output sequence is a DNA sequence. 
     
     
         36 . The method of any one of  claims 33 - 35 , wherein the first, second and/or third output sequence encodes one or more second output molecule. 
     
     
         37 . The method of  claim 36 , wherein the first and/or second output molecule controls a fifth heterologous promoter/operator controlling transcription of one or more fourth output sequence. 
     
     
         38 . The method of  claim 36  or  claim 37 , wherein the first and/or second output molecule is a protein. 
     
     
         39 . The method of any one of  claims 1 - 35 , further comprising culturing the cell under conditions that allow expression of the genetic circuit in the cell. 
     
     
         40 . A genetic circuit, comprising:
 (a) a first output sequence;   (b) a first promoter/operator controlling transcription of the first output sequence;   (c) a first guide RNA targeting the first promoter/operator;   (d) a second promoter/operator controlling transcription of the first guide RNA, wherein the second promoter/operator is input-sensitive such that a first input signal is required for induction of transcription of the first guide RNA;   (e) a first catalytically inactive endonuclease that in combination with the first guide RNA binds to a sequence targeted by the first guide RNA and prevents transcription of the first output sequence;   (f) a third promoter/operator controlling transcription of the first catalytically inactive endonuclease, wherein the third promoter/operator is input-sensitive such that a second input signal is required for induction of transcription of the first catalytically inactive endonuclease; and   (g) one or more heterologous polymerases that specifically bind one or more of the first, second and/or third promoter/operator.   
     
     
         41 . The genetic circuit of  claim 40 , wherein the genetic circuit further comprises a fourth promoter/operator controlling transcription of one or more second output sequence, wherein the fourth promoter/operator is input-sensitive such that a third input signal is required for induction of transcription of the one or more second output sequence. 
     
     
         42 . The genetic circuit of  claim 40  or  claim 41 , wherein the genetic circuit further comprises one or more endogenous polymerases that bind one or more of the first, second, third and/or fourth promoter/operator. 
     
     
         43 . The genetic circuit of  claim 41  or  claim 42 , wherein the third input signal is one or more second guide RNA encoded by the first output sequence and wherein the one or more second output sequence is one or more third guide RNA. 
     
     
         44 . The genetic circuit of any one of  claims 40 - 43 , wherein at least two of the first, second and third input signals are the same. 
     
     
         45 . The genetic circuit of any one of  claims 40 - 44 , wherein any of the first, second, third and/or fourth promoter/operators comprises a T7 promoter and an operator. 
     
     
         46 . The genetic circuit of any one of  claims 40 - 45 , wherein the heterologous polymerase is a viral polymerase. 
     
     
         47 . The genetic circuit of any one of  claims 40 - 46 , wherein the heterologous polymerase is a T7 RNA polymerase. 
     
     
         48 . The genetic circuit of any one of  claims 40 - 47 , wherein the genetic circuit further comprises polynucleotide sequences encoding one or more decoy operators having the same, or substantially the same, sequence as one or more of the operator sequences of the first, second, third and/or fourth promoter/operators. 
     
     
         49 . The genetic circuit of any one of  claims 40 - 48 , wherein the genetic circuit further comprises one or more polynucleotide sequences encoding one or more small RNAs (sRNAs) that binds to and sequesters the guide RNA. 
     
     
         50 . The genetic circuit of any one of  claims 40 - 49 , wherein the first, second, and/or third guide RNA is a nested guide RNA comprising two or more sequences that target two or more target sequences in the first, second, third and/or fourth promoter/operator, thereby causing promoter looping of the first, second, third and/or fourth promoter/operator. 
     
     
         51 . The genetic circuit of any one of  claims 40 - 50 , wherein the first, second and/or third input signal is a chemical, light, a polypeptide or a mechanical force. 
     
     
         52 . The genetic circuit of any one of  claims 40 - 51 , wherein the first, second and/or third input signal is isopropyl β-D-1-thiogalactopyranoside (IPTG), anhydrotetracycline (aTc), or 2,4-diacetylphloroglucinol (DAPG). 
     
     
         53 . The genetic circuit of any one of  claims 40 - 52 , wherein the first and/or second output sequence encode one or more first output molecule and the one or more first output molecule in turn becomes a fourth input signal required for controlling transcription of one or more third output sequences. 
     
     
         54 . The method of  claim 53 , wherein the first, second and/or third output sequence is a fourth guide RNA. 
     
     
         55 . The genetic circuit of any one of  claims 40 - 53 , wherein two or more input signals control transcription in a single promoter/operator. 
     
     
         56 . The genetic circuit of any one of  claims 40 - 55 , wherein the first catalytically inactive endonuclease is a RNA-guided DNA endonuclease. 
     
     
         57 . The genetic circuit of any one of  claims 40 - 56 , wherein the first catalytically inactive endonuclease is a catalytically inactive clustered regularly interspaced short palindromic repeat (CRISPR) endonuclease. 
     
     
         58 . The genetic circuit of any one of  claims 40 - 57 , wherein the first catalytically inactive endonuclease is catalytically inactive Cas9 or catalytically inactive Cpf1. 
     
     
         59 . The genetic circuit of any one of  claims 40 - 58 , wherein the first catalytically inactive endonuclease is selected from the group consisting of dSpCas9, dSpCas9(E), dSpCas9(VRER), dSpCas9(VQR), dSpCas9(EQR), desSpCas9, dSpCas9-HF1, dSaCas9, desSaCas9, dSt1Cas9, dFnCpf1, dAsCpf1, and dLbCpf1. 
     
     
         60 . The genetic circuit of any one of  claims 40 - 59 , wherein one or more nucleotide of the first, second, third and/or fourth guide RNA is mutated and the mutation of the first, second, third and/or fourth guide RNA does not decrease prevention of transcription of the first, second and/or third output sequence. 
     
     
         61 . The genetic circuit of any one of  claims 40 - 60 , wherein the first, second, third and/or fourth promoter/operator is sensitive to a guide RNA. 
     
     
         62 . The genetic circuit of any one of  claims 40 - 60 , wherein the first, second, third and/or fourth promoter/operator is sensitive to a chemical input. 
     
     
         63 . The genetic circuit of any one of  claims 40 - 62 , wherein the first, second, third and/or fourth promoter/operator comprises a polynucleotide sequence that encodes a T7 promoter and a polynucleotide sequence that encodes a PhlF operator. 
     
     
         64 . The genetic circuit of  claim 63 , wherein the T7 promoter and the PhIF operator control the transcription of guide RNA A2NT. 
     
     
         65 . The genetic circuit of  claim 64 , wherein the guide RNA A2NT controls the transcription of the A2NT operator. 
     
     
         66 . The genetic circuit of any one of  claims 40 - 65 , wherein one or more of the first, second and/or third output sequence is a DNA sequence. 
     
     
         67 . The genetic circuit of any one of  claims 40 - 66 , wherein the first, second and/or third output sequence encodes a second output molecule. 
     
     
         68 . The genetic circuit of  claim 67 , wherein the first and/or second output molecule controls a fifth heterologous promoter/operator controlling transcription of one or more fourth output sequences. 
     
     
         69 . The genetic circuit of  claim 67  or  claim 68 , wherein the first and/or second output molecule is a protein.